OBJECTIVE: To determine the real subcutaneous glucose concentration in healthy volunteers to help in the development of new calibration methods for subcutaneous glucosensors. RESEARCH DESIGN AND METHODS: We developed a new method to estimate the real subcutaneous glucose concentration based on the recirculation of phosphate-buffered saline (PBS) in a microdialysis probe inserted into the subcutaneous tissue. Tissue glucose diffuses into the probe until complete equilibration between the glucose concentration outside and inside the microdialysis probe is achieved. Later, the glucose content of the recirculated PBS is assessed in vitro. We applied the method in 10 healthy volunteers under fasting state and during a hyperglycemic clamp. In addition, we monitored the subcutaneous glucose with an enzymatic-amperometric glucosensor combined with a microdialysis probe. RESULTS: The subcutaneous glucose concentration measured by the recirculation method was 72 ± 6 and 78 ± 6% of the blood glucose measured in the fasting state and during the hyperglycemic clamps, respectively. On the other hand, the glucosensor's signal correlated significantly with the blood glucose. CONCLUSION: The recirculation method estimated the real subcutaneous glucose concentration, opening the way to develop new calibration procedures for subcutaneous glucosensors. However, a suitable calibration procedure is still lacking.